This invention relates generally to the field of quality control, and in particular to the quality control of a combinatorial library of compounds. More specifically, the invention relates to apparatus and methods for evaluating the quality of a library of compounds, typically produced by encoded split-pool synthesis.
The split-pool approach for solid phase synthesis is often used to rapidly create large, diverse chemical libraries of potential therapeutic compounds. This approach yields single beads of solid phase support to which a few hundred picomolecules of product compound are tethered. After synthesis, a pool of beads often can contain several thousand compounds.
It is desirable to verify the success of the synthesis before screening the library against biological targets. Although the library may be tested for activity in a high throughput screen in the event the library synthesis does not proceed as expected, the probability of identifying active compounds in this manner is very low. Even worse, an active component may turn out to be an unexpected (and irreproducible) side product.
Split pool synthesis is a method of producing a large number of compounds utilizing a minimal number of reaction steps in multiple reaction vessels. Exemplary split pool synthesis processes are described in U.S. Pat. Nos. 5,846,839 and 5,503,805; and in Kenneth C. Lewis et al., xe2x80x9cCharacterization of a Split Pool Combinatorial Library,xe2x80x9d LCGC, Vol. 16, No. 7, pp. 644-649, July 1998; and Z. Ni et al., xe2x80x9cA Versatile Approach To Encoding Combinatorial Organic Synthesis Using Chemically Robust Secondary Amine Tags,xe2x80x9d J. Med. Chem., 39:1601-1608 (1996). The complete disclosures of all of these references are herein incorporated by reference.
Merely by way of example, one exemplary synthesis process proceeds by attaching a photo cleavable linking compound to 90% of the amines on the bead surface while protecting the remaining amino groups with N-(allyloxycarbonyl)(Alloc). The beads are split into 35 pools and the first building block is attached to the photo-linking compound. Dailkylamine tags for the first building block are attached to the 10% residual amines. The beads are pooled and resplit to attach the second building block. Dailkylamine tags for the second building block are attached, and the beads are pooled and resplit to attach the third building block set to the scaffold. The 35 samples are then stored as separate subpools of the library. In this way, each resulting bead contains a desired compound and a series of dailkylamine tags that serve as a record of the synthesis procedure.
The quality control of split pool libraries presents a variety of challenges to analytical chemists. First, such libraries provide a limited amount of compound. For example, the actual amount of compound can vary from less than 50 pmol to more than 1 nmol depending on the size and type of bead used as the solid support. Merely by way of example, some processes may utilize 130 xcexcm dp Tentagel beads (Nova Biochem, San Diego, Calif.), which can be loaded to approximately 400 pmol/bead. However, the actual loading on such beads is often less due to the variations in bead size, synthetic yield and efficiency of cleavage.
Another set of problems occurs after the sample is separated from the bead. Such problems can include those relating to contamination, adsorption, and the transfer of small volumes. Because the sampling of a split pool library is random, chemists must analyze enough beads to obtain meaningful statistics about the library. Therefore, the analysis method must handle limited quantities of sample in a high throughput manner.
Hence, it would be desirable to provide devices and methods to facilitate the evaluation of the quality of a library of combinatorial compounds. Such devices and methods should be able to handle limited quantities of sample in a high throughput manner. As such, the devices and methods should be compatible with existing analysis and fluid handling equipment. Further, the devices and methods should be efficient to reduce the time required to evaluate the quality of the library.
The invention provides exemplary devices and methods for evaluating the quality of a combinatorial library of compounds. One exemplary device comprises a holding plate having an array of apertures. A plurality of vials are removably held within the apertures, with each vial having an open top end. A seal member is disposed over the top ends of the vials, and a top plate is removably coupled to the holding plate to force the seal member against the top ends of the vials. Such a device is particularly advantageous in that it provides a way to keep the beads in a sealed environment during both ligand cleavage and tag cleavage. In this manner, the beads do not need to be transferred to separate vials during a quality control process. For example, following ligand cleavage, the tags are typically released for analysis by acid hydrolysis. This step typically requires the beads to be heated. By utilizing the device of the invention, the beads may remain within the same vials used for ligand cleavage, and simply placed in an oven during the hydrolysis process.
Conveniently, the device may further include a bottom plate having a cavity for receiving bottom ends of the vials. Preferably, the bottom plate has a standard footprint to allow the device to be utilized with automated equipment, such as autosamplers. In a similar manner, the apertures in the holding plate are preferably spaced apart to correspond to a standard multi-well plate format, e.g., the 96-well format. In this manner, automated equipment may also be used to introduce and remove fluids from the vials. In this manner, the time to perform the quality control process is greatly reduced.
The top plate and the holding plate are preferably constructed of a rigid material that is resistant to acids. For example, the top plate and holding plate may be constructed of an anodized aluminum. Conveniently, a plurality of screws may be provided to removably couple the top plate to the bottom plate. In still another aspect, the seal member preferably comprises a resilient sheet having the same dimensions as the holding plate. The resilient sheet preferably includes a layer of PTFE and the vials are preferably constructed of glass so that the sheet and the vials will be compatible with the ligand and tag cleavage processes.
In still yet another aspect, the top plate includes a plurality of through holes which are aligned with the vials when the top plate is coupled to the holding plate. In this way, the seal member may be pierced and fluids within the vials extracted through the through holes. Optionally, a resilient pad may be disposed adjacent the holding plate and has openings which correspond to the apertures of the holding plate. The resilient pad is useful in protecting the vials from breaking when the device is assembled.
The invention further provides an exemplary method for evaluating the quality of a combinatorial library of compounds. According to the method, a device is provided which comprises a holding plate and a plurality of vials that are held by the holding plate. At least one solid support is placed into at least some of the vials. Each solid support has a ligand and encoded tags. The solid supports are then sealed within the vials. Once sealed, the ligand is cleaved from the solid supports, and a ligand sample is removed from the vials. Following the ligand cleavage, the encoded tags are cleaved from the solid supports. To cleave the encoded tags, the vials are preferably heated while the solid supports are sealed within the vials. Samples of the cleaved tags are then removed from the vials. The ligand is then compared to a structure indicated by the tags for each solid support.
To seal the solid supports within the vials, a seal member is preferably placed over the vials and a top plate is coupled to the holding plate to force the seal member against the vials.
In one particular aspect, a photo-cleavage process is employed to cleave the ligands from the solid support. Conveniently, the vials may be turned upside-down and then placed under a lamp to photo-cleave the ligands. When the vials are turned upside-down, the solid supports remain within the bottom ends of the vials due to the surface tension of the fluids with the vials.
In another aspect, the top plate has through holes which are aligned with the vials. In this manner, the seal member is pierced to extract the ligand sample from the vials.
In yet another aspect, the encoded tags are cleaved by first removing the top plate and the seal member and introducing a cleaving solution, such as hydrochloric acid, into the vials. A fresh seal member is then placed over the vials and the top plate is replaced to seal the solid supports within the vials. Once properly sealed, the device is transferred to an oven where the vials are heated. Following heating, the top plate and the seal member are preferably removed to allow the cleaving solution to be dried. For example, the device may be placed in a vacuum oven to dry the hydrochloric acid.
Following drying of the cleaving solution, a solvent, such as acetonitrile is introduced into the vials. The top member and the seal member are then replaced. To remove the cleaved tags from the vials, the seal member is pierced and a sample is extracted. Preferably, the ligand sample and the samples of the cleaved tags are evaluated using mass spectrometry. These results are then compared to determine the quality of the combinatorial library of compounds.